J. Med. Chem. 1988,31,122-129
122
2,4-Diamino-5-benzylpyrimidines and Analogues as Antibacterial Agents. 9. Lipophilic Trimet hoprim Analogues as Antigonococcal Agents' B. Roth,*t D. P. Baccanari, C. W. Sigel, J. P. Hubbell, J. Eaddy, J. C. Kao, M. E. Grace, and B. S. Rauckman Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709. Received June 15, 1987 Lipophilic analogues of trimethoprim (1) bearing 3,5-dialkyl-4-hydroxysubstituents in the benzene ring are much more active in vitro against Neisseria gonorrhoeae than is 1. The 3,5-diisopropyl-4-hydroxy derivative (2) was selected as a candidate for clinical evaluation as an antigonococcal agent, and as part of the preliminary evaluation it was submitted to extended pharmacokinetic and metabolism studies in dogs. Although the compound was not extensively conjugated by metabolic enzymes, one of the methyl groups was metabolized t o produce a 3-isopropyl-4hydroxy-5-(a-carboxyethyl)benzylderivative (43), which was rapidly excreted. Related analogues were likewise extensively metabolized. In paper 7 of this series,2we described the activities of a series of trimethoprim (TMP) (1) analogues bearing alkyl substituents in the 3- and 5-positions of the benzene ring and alkoxy, hydroxy, or amino functions in the 4-position against four dihydrofolate reductase (DHFR) isozymes. Some of these compounds were found to be much more active than trimethoprim against Neisseria gonorrhoeae DHFR,particularly derivatives with n-propyl or isopropyl substituents a t the 3- and 5-positions. This paper is focused largely on the 3,5-dialkyl-4-hydroxybenzyl derivatives as potential antigonococcal agents, with particular emphasis on the 3,5-diisspropyl-4-hydroxybenzyl derivative (2).
HZN
$r-y(R3 R4
R5
1 2
m e t h y l l p h e n ~ lwe , ~ decided to pursue this approach further, as well as the coupling of 5-(hydroxymethy1)uracil with 2,6-disubstituted phenols. Scheme I illustrates the coupling of 3 with 4, compared with the reaction of 5 plus 6. Both reactions proceeded satisfactorily to give 7, but the Mannich procedure gave a far superior yield. Chlorination of both 7 and 8 gave good yields of 9 and 10. Amination proceeded in excellent yield with 10 to produce 2, but under similar conditions the di-tert-butyl analogue was only partially diaminated, for reasons that are not clear. Further attempts were made to produce 11 from 12 and 4, which finally resulted in achievement of a 10% yield of the desired product, along with 5% of 13, in which one of the tert-butyl groups was lost. No further attempts were made to improve the route to 11, since 2 proved a considerably more interesting target. Scheme I1 illustrates the route used to obtain radiolabeled 2, which required additional steps, but was highly satisfactory. Biological Data A. In Vitro Antibacterial Activities. Table I lists the in vitro antibacterial activities of (3,5-dialkyl-4hydroxybenzyl)- and (3,5-dialkyl-4-methoxybenzyl)pyrimidines against 10 selected organisms, relative to that of trimethoprim (1). In most cases, serial dilutions were carried out a t levels of 0.1, 0.3, 1, 3, etc., and differences of fl dilution are not significant. In practically every case the compounds were significantly less active than 1, particularly against Gram-negative organisms. This is very often the case with analogues of 1 that are more lipophilic.6 Compounds 25 and 27 were nearly equipotent with 1, and several of the compounds were very active against such Gram-positive organisms as Staphylococcus aureus and Streptococcus faecium (not listed here). However, none could be considered outstanding for the standard series of organisms tested. The compounds most active against Escherichia coli DHFR had been found to be the 3ethyl-5-n-propyl-4-hydroxy and 3,5-di-n-propyl-4-hydroxy derivatives 27 and 28.2 Compound 27 is the most active of these against whole cells.
R 3 I R4, R5 = OMe (Trmethoprim) R 3 , R5, = Prd,
R 4 = OH
General in vitro antibacterial screening results on the more promising compounds of paper 7 are detailed here, along with data on selected compounds in a more specific in vitro antigonococcal screen. No convenient animal model is available for assessing activity against this org a n i ~ m . Therefore, ~ several of the most promising leads were selected for extended pharmacokinetic and metabolic studies in rats and dogs to assess their potential utility, the results of which are described here, as are some improved synthetic methods. Chemistry In paper 6 of this ~ e r i e swe , ~ described the acid-catalyzed condensation of 2,6-dialkylphenols with 2,4-diamino-5(hydroxymethy1)pyrimidine to yield 2,4-diamino-5-(3,5dialkyl-4-hydroxybenzy1)pyrmidinesin a single-step condensation. Although this reaction worked reasonably well with lower alkyl derivatives, it produced only 20% of 2 and only traces of the di-tert-butyl analogues under the conditions described. At an earlier date, we had prepared the 3,5-diisopropyl and di-tert-butyl-4-methoxy derivatives in overall yields of 16-38% from phenolic Mannich condenfob sations with 2,4-diamino-6-(methylthio)pyrimidine, lowed by alkylation of the phenol and d e t h i a t i ~ n . ~Although this method was successful, it was lengthy, and the Raney nickel dethiation was somewhat erratic. We never tried to make the phenolic 6-unsubstituted pyrimidines by this procedure. Since we had found that uracil reacted successfully with 2,6-diisopropyl-4-[(dimethylamino)Present address: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514.
(1) Roth, B.; Baccanari, D. P.; Sigel, C. W.; Hubbell, J. P. Abstracts of the 6th International Symposium on Future Trends in Chemotherapy, Tirrenia (Pisa), May 1984, p 170. (2) Roth, B.; Rauckman, B. S.; Ferone, R.; Baccanari, D. P.; Champness, J. N.; Hyde, R. M. J . Med. Chem. 1987,30,348. (3) DiGiacomo, R. F.; Gale, J. L.; Holmes, K. K.; Buchanan, T. M. Infect. Immun. 1977, 15, 670. (4) Stuart, A.; Paterson, T.; Roth, B.; Aig, E. J . Med. Chem. 1983, 26, 667. (5) Roth, B.; Aig, E.; Lane, K.; Rauckman, B. S. J . M e d . Chem. 1980,23, 535. (6) Roth, B.; Aig, E.; Rauckman, B. S.; Strelitz, J. Z.; Phillips, A. P.; Ferone, R.; Bushby, S. R. M.; Sigel, C. W. J . Med. Chem. 1981,24, 933.
0022-2623/88/ 1831-0122$01.50/0 0 1987 American Chemical Society
Lipophilic Trimethoprim Analogues
Journal of Medicinal Chemistry, 1988, Vol. 31, No. 1 123
Table I. Comparative in Vitro Antibacterial Activity of (3,5-Dialkylbenzyl)pyrimidinesand Analogues against Selected Organisms vs. Trimethoprim Standard (MIC Compound/MIC TrimethoprimIa organismb benzene Staph. Vibrio Myco. Shig. Serr. Kleb. Entero. Proteus E. coli marc. pneum. aerog. vulgaris no. 3 4 5 aureus chol. smeg. Sal. typh. flex. 1 OMe OMe OMe 1 1 1 1 1 1 1 1 1 1 2 Pr-i OH Pr-i 10 100 300 300 100 300 10 >loo0 >loo0 >loo0 >loo >loo0 >loo0 >loo0 11 Bu-t OH Bu-t 30 24 Me OH Me 10 10 10 3 1 3 25 Me OH E t 1 1 3 3 3 1 3 3 1 26 Et OH E t 3 1 3 10 10 3 10 10 10 10 27 Et OH Pr-n 0.3 1 3 3 3 10 0.3 3 10 3 28 Pr-n OH Pr-n 0.3 1 3 10 10 10 0.3 30 30 30 29 Me OH Bu-t 3 1 100 '30 100 3 100 100 100 30 Me OMe Me 100 100 1000 300 1000 300 31 Me OMe Et 3 3 3 30 30 3 30 30 3 32 Et OMe Et 0.3 3 3 3 10 10 30 33 Et OMe Pr-n 0.3 10 3 30 30 30 1 30 100 30 34 Pr-n OMe Pr-n 1 10 1 100 100 30 1 100 100 >30 35 Pr-i OMe Pr-i 30 3000 3000 >loo0 >loo0 >loo0 36 Me OMe Bu-t 10 10 10 300 300 100 10 >100 300 >30 37 Bu-t OMe Bu-t 30 3000 10000 >loo0 >loo0 >loo0 " MIC = minimal inhibitory concentration, determined as micrograms per milliliter. Numbers greater than one indicate less activity for the test compound than for trimethoprim. Typical MIC values (pg/mL) for 1 with the organisms listed are 0.3-1, 0.3-1, 1-3, 0.03-0.1, 0.1-0.3, 0.1-0.3, 10, 0.03-1.0, 0.1-0.3, and 1-10. *Staphylococcus aureus CN 491, Vibrio cholerae ATCC 14035, Mycobacterium smegmatis S3254, Salmonella typhosa CN 512, Shigella flexneri CN 6007, Escherichia coli CN 314, Serratia marcescens CN 2398, Klebsiella pneumoniae CN 3632, Enterobacter aerogenes 2200/186, Proteus vulgaris CN 329.
Table 11. Comparison of Inhibitory Activities of Selected 3,4,5-Substituted (Alkoxy-, Alkyl-, and Alkenylbenzy1)pyrimidines against Neisseria gonorrhoeae DHFR with Their in Vitro Antibacterial Activities (MIC) against Neisseria gonorrhoeue benzene subst MIC, /IM, vs IbO,M, X lo8 vs N. N. gonorrhoeae 5 gonorrhoeae DHFR" compd no. 3 4 9.2 2.9 Pr-i OH Pr-i 2 4.4 17.5 Et OMe Pr-n 33 9 4.9 Et OH Pr-n 27 5.8 13.0 Bu-t OMe Bu-t 37 6.0 20 Pr-n OMe Pr-n 34 6.1 35 Bu-t OH Bu-t 11 6.7 54 OMe OMe Pr-n 3Sb 110 CH=CHMe 9.3 OMe OMe 39b 110 CHZCH=CHz 11 OMe OMe 40b 17 180 OMe OMe OEt 41b 31 140 Me OMe Et 31 37 200 Me OMe Me 30 1 OMe OMe OMe 45 240 aSee ref 2 for methodology. *Roth, B.; Tidwell, M. Y.; Ferone, R.; Baccanari, D.; Sigel, C.; Elwell, L., manuscript in preparation. Table 111. Compounds Chosen for Pharmacokinetic Studies and Their Inhibitory Activities against Neisseria gonorrhoeae and Rat Liver DHFR, Compared to 1 benzene subst I,,, M, X lo8 vs DHFR compd no. 3 4 5 N. gonorrhoeae Rut Liver 2 Pr-i OH Pr-i 2.9 720 11 Bu-t OH Bu-t 6.1 3450 26 Et OH Et 12 1600 33 Et OMe Pr-n 4.4 3000 37 Bu-t OMe Bu-t 5.8 3100 42" Pr-n OH Allyl 2.0 363 1 OMe OMe OMe 45 34000 " See footnote b, Table 11.
Initial screening for N. gonorrhoeae wm carried out with clinical isolates. Among a very large series of compounds tested, the only ones showing activity superior to 1 were the lipophilic (3,5-dialkylbenzyl), (3,4-dimethoxy-5-a1kylbenzyl, or -alkenylbenzyl), or (3,4-dimethoxy-5-aralkylbenzy1)pyrimidines. The latter series will be discussed in a separate paper. A subset of the most active compounds of Table I, as well as compounds 38-41, were selected for careful comparison in a separate test against a single strain of N. gonorrhoeae (F62). These results are shown in Table 11. The MIC data are compared with inhibitory activities against DHFR from the same strain, and it will be observed that the relative activities are very
well correlated. The most active compounds are 2 and 27; 2 is 15 times better as a gonococcal enzyme inhibitor than 1, with 27-fold greater in vitro antigonococcal activity. B. Pharmacokinetic Studies in Dogs. The concentrations in blood, urine, and tissues of mice after oral administration were determined for several compounds showing significant antigonococcal activity (S. R. M. Bushby, unpublished results). Six compounds were then chosen for further pharmacokinetic study in the dog, on the basis of all previous screening data. These are shown in Table 111. Since the most active compounds were the 4-hydroxy derivatives, consideration was also given to the relative probabilities of glucuronide or sulfate formation
124 Journal of Medicinal Chemistry, 1988, Vol. 31, No. 1
Roth et al.
Scheme I
OH 0
Bu-t
0
OH
Bu-t
0
2 +
Me2NCH2+oH
H
P
-7 R,,R,= 8 R,, R,
h
Bu-t
% Rg , R,
= Pr-i
Le R,, R,
=
Bu-t
11R 3 , R,
= Pr.i
=
2R3,R,=
“2
Bud + other products ( 1 3 l5)
Pr-i
”2
Scheme I1
tetramine
Pr-i hexamethylene-
i.pr+
pr.i
HC(OMe)3
i-Pr+
CH(OMe),
C HO
fi
1L
Pr-i
19.
I
1) NaH, PhCH,Br 2) HCI
OCH2 Ph
Pr-i
Pr4
i-pr*
2p CHO
Pr-i
*2c -2;L
in vivo, which would lead to rapid excretion. Trimethoprim itself is metabolized in part to a 4-hydroxybenzyl derivative, which is rapidly converted to a glucuronide, followed by rapid e ~ c r e t i o n .Flanking ~ of the 4-hydroxy substituent by branched-chain alkyl substituents might be expected to minimize such enzymatic inactivation, but (7) Sigel, C. W. in Handbook of Experimental Pharmacology; Hitchings, G. H., Ed.;Springer-Verlag: Heidelberg, 1983; Vol. 64, pp 164-184.
a comparison of branched and straight-chain analogues was considered essential. The dog was chosen for the more extended studies, since from previous data with analogues of 1, the dog kinetic data correlated better with human results than did other laboratory ani mal^.^ For oral absorption studies, four beagle dogs (two males and two females) received single doses (5 mg/kg) of each drug in a gelatin capsule. Only very low concentrations of compounds 11, 37, and 42 were observed in serum
Journal of Medicinal Chemistry, 1988, Vol. 31, No. 1 125
Lipophilic Trimethoprim Analogues
Table JV. Tissue Concentrations (pglg) in Male Rats, Ovary Concentrations (wg/g) in Female Rats, and Serum Concentrations (gg/mL) in Male and Female Rats of Compound 2 (Equivalents) at Various Times following Oral Administration of 2-14C-2 (Average of Three Rats) time after serum dose, h stomach intestine liver kidney lung muscle brain testes ovary male female 0.25 386.97 f 114.44 0.50 303.84 f 103.15 0.75 319.16 f 51.06 1.00 46.97 f 15.90 1.50 97.08 f 70.22 2.00 46.08 f 32.77 3.00 8.92 f 5.71 4.00 14.75 f 10.14 6.00 5.22 f 5.39 10.00 2.00 f 1.54 24.00 1.49 f 2.40
157.50 f 61.60 114.48 i 27.89 112.23 f 85.55 25.97 f 11.09 14.91 f 7.75 11.59 f 3.83 7.34 f 1.47 5.57 f 1.38 4.13 f 1.50 2.29 f 1.33 0.85 f 1.24
17.42 f 1.70 29.9 f 6.35 23.13 f 2.06 13.51 f 1.58 13.46 f 0.45 10.88 f 0.97 9.56 f 0.95 8.23 f 1.27 5.14 f 1.05 5.67 f 0.97 2.71 f 0.94
2.98 f 1.59 8.43 f 1.45 10.29 f 0.19 7.78 f 1.79 6.74 f 0.63 4.08 f 0.71 4.11 f 0.15 2.07 f 0.66 1.55 f 0.26 1.13 f 0.51 0.50 f 0.32
2.52 f 1.35 8.31 f 3.41 11.33 f 0.38 11.88 f 2.26 7.17 f 0.22 4.98 f 2.45 4.18 f 0.92 2.30 f 1.63 0.52 f 0.11 0.40 f 0.27 0.10 f 0.08
0.44 f 0.29 1.45 f 0.87 2.31 f 0.84 2.36 f 0.47 1.84 f 0.23 1.40 f 0.40 1.38 f 0.29 0.67 f 0.43 0.25 f 0.05 0.13 f 0.04 0.04 f 0.01
0.13 f 0.05 0.58 f 0.13 1.01 f 0.10 0.91 f 0.20 0.65 f 0.12 0.43 f 0.23 0.30 f 0.03 0.19 f 0.15 0.05 f 0.01 0.04 f 0.02 0.004 f 0.003
1.34 f 0.29 1.07 f 0.44 1.77 f 0.34 1.32 f 0.25 1.52 f 0.21 1.24 f 0.43 1.79 f 0.19 1.01 f 0.56 0.41 k 0.07 0.66 f 0.72 0.15 f 0.03
2.06 f 1.06 4.06 f 2.18 4.48 f 0.78" 3.33 f 0.78" 4.50 f 0.83 1.93 f 0.57 2.06 f 1.09 1.55 f 0.13 0.71 f 0.56 0.23 f 0.09 0.08 f 0.06
0.45 f 0.21 1.12 f 0.29 1.13 f 0.13 0.92 f 0.08 0.80 f 0.08 0.49 f 0.04 0.47 f 0.08 0.42 f 0.14 0.27 f 0.08 0.23 f 0.09 0.08 f 0.06
0.49 f 0.12 0.90 f 0.17 1.44 f 0.30 1.09 i 0.42 1.09 f 0.15 0.68 f 0.17 0.61 f 0.20 0.53 f 0.12 0.34 f 0.18 0.16 f 0.06 0.14 f 0.04
Average of two rats.
samples (
